Objective: The objective of this study was to investigate the correlation between in vivo d-tocotrienol (DT3) pharmacokinetics, pharmacodynamics and radiation protection, and to evaluate the effect of DT3 pre-treatment on radiation-induced alterations in apoptotic and autophagic pathways. Methods: We evaluated pharmacokinetics (plasma, 0.5 to 12 h) and pharmacodynamics (peripheral blood indices; day 3, 7, 10 and 14) after a single subcutaneous injection of 300 mg kg -1 DT3 in unirradiated CD2F1 mice. Next, we monitored 30-day post-irradiation survival (9.25 Gy) and haematopoietic recovery of DT3-treated mice (7 Gy) exposed to cobalt-60 c-irradiation. The effects of DT3 on irradiated bone marrow apoptosis and autophagy were determined by analyses of key caspases (3, 7, 9 and 8), beclin-1 and light chain 3 conversion. Results: Plasma concentration of DT3 reached ,195 mM (Cmax) 1 h after injection (Tmax), and DT3 was eliminated from plasma 12 h later. In unirradiated mice, DT3 significantly increased white blood cells (WBCs), neutrophils, lymphocytes (day 3 post DT3 injection) and platelets (day 7) by 1.5-to 2-fold, over vehicle-treated control. DT3 pre-treatment improved 30-day survival to 100% (,15% in control) and accelerated recovery of reticulocytes, platelets, WBCs, neutrophils, lymphocytes and monocytes in peripheral blood. DT3 reduced activation of caspase-8, caspase-3 and caspase-7, inherent to apoptosis, while increasing autophagy-related beclin-1 expression in irradiated bone marrow. Conclusion: These data indicate that DT3 stimulates multilineage haematopoiesis, protects against radiation-induced apoptosis downstream of the mitochondria and stimulates cytoprotective autophagy. Apart from a potent antioxidant activity, DT3 may elicit survival advantage following irradiation by enhancing haematopoiesis and modulating signalling pathways.
Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 ( SMN1 ) transgene (AAV-PHP.B-CBh- SMN1 ) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 10 13 , 5 × 10 13 , or 1 × 10 14 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 10 13 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.
ALXN4100TPO, a thrombopoietin (TPO) receptor agonist, increases platelets, abrogates radiation-induced thrombocytopenia and affords significant survival benefit to lethally irradiated mice. This preliminary nonclinical safety study assessed effects of a single subcutaneous (sc) administration of ALXN4100TPO in CD2F1 mice randomized into naïve, control antibody (ALXN4200, 100 mg/kg), low (1 mg/kg), medium (10 mg/kg), or high (100 mg/kg) ALXN4100TPO doses. End points included clinical observations, body weight changes, hematology, histopathology, pharmacokinetics, pharmacodynamics by measuring platelet counts, and endogenous TPO (eTPO) levels. Salient findings were prominent increase in platelet counts and end cells of myeloid and lymphoid lineages; elevated megakaryopoiesis in bone marrow; and extramedullary hematopoiesis in spleen and liver. Serum ALXN4100TPO levels were maximum 24 hours after administration, with a half-life of 13 days. Endogenous TPO levels were elevated in 10 and 100 mg/kg ALXN4100TPO-treated groups. In conclusion, ALXN4100TPO (1-100 mg/kg, sc) treatment in CD2F1 mice resulted in profound pharmacological changes in the hematopoietic tissue; however, no life-threatening adverse events were observed.
FXaI16L is a variant of activated coagulation factor Xa in which the conformational transition from zymogen to active protease is impaired. Binding to FVa facilitates its transition to the active conformation, rescues procoagulant activity and is hypothesized to localize FXaI16L hemostatic effect to sites of hemorrhage. In nonclinical toxicology studies, thrombi/emboli were observed in animals administered doses exceeding the projected efficacious dose. To identify a possible biomarker predictive of FXaI16L -related thrombi/emboli, effects of FXaI16L on platelets (PLAT), fibrinogen (FBGN), activated partial thromboplastin time (aPTT), prothrombin time (PT), factor V activity (FV), protein C (PROT C) activity, tissue factor pathway inhibitor (TFPI), and antithrombin III (AT-III), were evaluated in in male cynomolgus monkeys (3/group) given FXaI16L for 5 days at IV bolus dosages of 0, 0.1, or 1 mg/kg/day (administered as 0, 0.05, 0.5 mg/kg/dose; BID, 8 h apart). Changes in these parameters were evaluated 5 and 30 min after each dose on Days 1, 3 and 5, and histological evaluation for thrombi/emboli occurred approximately 45 min after first daily dose on Day 5. A single thrombus was observed in the heart of a single animal given 0.1 mg/kg/day, multiple thrombi/emboli were observed in the heart, lung and/or kidney of all animals given 1 mg/kg/day. Compared with pretest values, decreased (range) PLAT (24%-45%), FBGN (11%-86%), aPTT (11%-30%), FV (11%-76%), PROT C (60%-100%), ATIII (2%-26%), and prolonged PT (5%-524%) were observed at ³0.1 mg/kg/day, and prolonged aPTT (17%-367%) and increased TFPI (10%-49%) occurred at 1 mg/kg/day. PROT C activity was below detectable levels 5 min after dosing 1 mg/kg/day, a dose that caused multiple thrombi/emboli. Thus, PROT C activity (as measured by StaClot activity assay) may have utility as a safety biomarker for monitoring treatment with FXaI16L. Depletion of PROT C activity in the 5-day biomarker study in monkeys may reflect actual depletion of PROT C, or may reflect pharmacologic activity of FXaI16L that interferes with the clot based endpoint of this assay since the compound may inherently decrease clotting times. To distinguish between these possibilities, in vitro experiments were performed wherein varying concentrations of FXaI16L were added to normal human plasma and specimens were assayed by the StaClot® (clot based)PROT C activity assay, the StaChrome® (chromogenic) PROT C activity assay and by the Asserachrom® (ELISA) PROT C assay. In a similar manner, effect of FXaI16L on protein S levels was assessed using the StaClot® (clot based) protein S activity assay and an antigenic assay for free protein S. Following in vitro incubation of human plasma with increasing concentrations of FXaI16L, clot-based methods for PROT C and Protein S showed dose-dependent decreases in activity. In contrast, chromogenic and ELISA-based methods for PROT C or antigenic assay for Protein S remained unchanged from baseline levels, confirming that FXaI16L interferes with the StaClot® assay and does not deplete Protein S or PROT C. Abstract 4237. Table 1Comparison of PROT C and Protein S in Human Plasma When Incubated In Vitro with FXaI16L and Assessed by Clot-based or Antigenic AssaysAssay NameIncreasing Concentrations of FXaI16L0 ng/mL1 ng/mL5 ng/mL25 ng/mL50 ng/mL100 ng/mLStaClot PROT Ca137.5 ± 17.37132.8 ± 19.75107.5 ± 24.3457.0 ± 31.01*27.0 ± 19.82*5.3 ± 6.18*Stachrom PROT C122.0 ± 22.73121.3 ± 20.48121.0 ± 22.05121.5 ± 22.41121.5 ± 23.23119.8 ± 21.70Asserachrom PROT C85.8 ± 9.1891.8 ± 11.2790.8 ± 13.4389.0 ± 12.1185.3 ± 14.5286.3 ± 14.38StaClot Protein Sa86.4 ± 21.5766.0 ± 19.6514.6 ± 18.50*0.0 ± 0.00*0.0 ± 0.00*0.0 ± 0.00*Liatest Free Protein S103.6 ± 28.34106.6 ± 28.51100.0 ± 26.67103.0 ± 27.06102.2 ± 29.0399.2 ± 25.35 Data presented as Mean ± S.D. n = 5 donor plasmas/drug concentration. aStaClot PROT C or Protein S – clot-based endpoint. *Statistically different from baseline (0 ng/mL). P<0.05. Conclusion: Pharmacologic activity of FXaI16L interferes with the clot-based PROT C and protein S assays. However, because marked decreases in the clot-based PROT C assay correlated with the observation of thrombi/emboli in monkeys, this assay remains a useful safety biomarker for monitoring treatment with FXaI16L. Regardless, the impact of drug pharmacology on the respective assay mechanism should be considered when interpreting results. Disclosures Bolt: Pfizer: Employment. Hua:Pfizer Inc: Employment. Brenneman:Pfizer: Employment. Graves:Pfizer: Employment. Arkin:Pfizer Inc.: Employment. Criswell:Pfizer: Employment.
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